Biological assays have provided a valuable tool in the evaluation of the effect of pharmacologically active compounds including biologically active proteins such as interferons, interleukins, B-cell growth factor or platelet derived growth factor on the growth and viability of a selected cell population. Procedures developed for the bioassay of these types of molecules are usually biphasic. The initial phase consists simply of incubating the substrate cell population with the substance to be tested for a desired period of time. The art has developed several divergent procedures to carry out the second phase of the bioassay, the quantitative analysis of the cellular response to the sample compound.
Generally, the assays of the art include immuno-mediated assays which are rapid but quantitate only the binding of the molecules to the cell. Another type of assay requires cell staining and visual or microscopic observation of a change in cellular morphology. Such assays are dependent on a subjective evaluation of the results. Still another type of assay system involves use of radioactive metabolites to determine viability. This system is less subjective than the colormetric systems previously described but the processing of numerous samples is time consuming, labor intensive and expensive. Counting the assay cells with the aid of an electronic particle counter to determine cell growth requires large multiples of each sample for accurate results and does not truly reflect the viability of the assay cells.
More recently, colorimetric analysis of cell growth, either by direct staining of a cell monolayer or by cellular dye uptake have been developed. These procedures are less objective then most methods of determining cell growth and many of the steps of the assays are readily adaptable to automation.
An example of one such recently developed system is described by Yeh et al., Journal of Clinical Microbiology, 16, No. 2, 413-415 (1982). In this assay methodology involving a cytopathic effect reduction assay for human interferon, interferon titers were calculated from optical density readings of crystal violet-stained monolayers in an automated spectrophotometer. Multiple samples are handled in such system by utilizing a 96-well microtiter plate.
Another system is described in the paper by Klebe in In Vitro 20, No. 2, 127-132 (1984). In this system nonlethal vital stains comprising two tetrazolium stains identified as MTT and IN tetrazolium are employed. This system is utilized to identify clones derived by genetic mutation or from cell fusions such as for example in the case of producing antibodies secreting hybridomas.
In yet another system described by Zolg et al. in In Vitor, 20 No. 3, 205-215 (1984) lactic acid is used as a metabolic marker for the growth of the desired cell population. This system was utilized to optimize growth of malarial parasites for use in studying this disease. The authors observed that the production of lactic acid by the parasitic cells was dependent upon the developmental stage of the parasites. Detection of the lactic acid obtained from the cell population was accomplished using a commercial lactate test kit involving detecting absorbance at 365 nm of the assay solution.